Isoxazole Carboxamides as Irreversible SMYD Inhibitors

ABSTRACT

The present disclosure provides provides substituted isoxazole carboxamides having Formula (I), and the pharmaceutically acceptable salts and solvates thereof, wherein R 1 , R 2a , R 2b , R 3a , R 3b , X, n, and m are defined as set forth in the specification. The present disclosure is also directed to the use of compounds of Formula I to treat a disorder responsive to the blockade of SMYD proteins such as SMYD3 or SMYD2. Compounds of the present disclosure are especially useful for treating cancer.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure provides substituted isoxazole carboxamides asSMYD protein inhibitors, such as SMYD3 and SMYD2 inhibitors, andtherapeutic methods of treating conditions and diseases whereininhibition of SMYD proteins such as SMYD3 and SIVIYD2 provides abenefit.

Background

Epigenetic regulation of gene expression is an important biologicaldeterminant of protein production and cellular differentiation and playsa significant pathogenic role in a number of human diseases. Epigeneticregulation involves heritable modification of genetic material withoutchanging its nucleotide sequence. Typically, epigenetic regulation ismediated by selective and reversible modification (e.g., methylation) ofDNA and proteins (e.g., histones) that control the conformationaltransition between transcriptionally active and inactive states ofchromatin. These covalent modifications cart be controlled by enzymessuch as methyltransferases (e.g., SMYD proteins such as SMYD3 andSMYD2), many of which are associated with genetic alterations that cancause human disease, such as proliferative disorders. Thus, there is aneed for the development of small molecules that are capable ofinhibiting the activity of SMYD proteins such as SMYD3 and SMYD2.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides substituted isoxazolecarboxamide compounds represented by Formulae I-XII below, and thepharmaceutically acceptable salts and solvates thereof, collectivelyreferred to herein as “Compounds of the Disclosure.”

In another aspect, the present disclosure provides a Compound of theDisclosure and one or more pharmaceutically acceptable carriers.

In another aspect, the present disclosure provides a method ofinhibiting SMYD proteins, such as SMYD3 or SMYD2, or both, in a mammal,comprising administering to the mammal an effective amount of at leastone Compound of the Disclosure.

In another aspect, the present disclosure provides a method ofirreversibly inhibiting SMYD proteins, such as SMYD3 or SMYD2, or both,in a mammal, comprising administering to the mammal an effective amountof at least one Compound of the Disclosure.

In another aspect, the present disclosure provides methods for treatinga disease, disorder, or condition, e.g., cancer, responsive toinhibition of SMYD proteins, such as SMYD3 or SMYD2, or both, comprisingadministering a therapeutically effctive amount of a Compound of theDisclosure.

In another aspect, the present disclosure provides the use of Compoundsof the Disclosure as inhibitors of SMYD3.

In another aspect, the present disclosure provides the use of Compoundsof the Disclosure as inhibitors of SMYD2.

In another aspect, the present disclosure provides the use of Compoundsof the Dicslosure as inhibitors of SMYD proteins.

In another aspect, the present disclosure provides a pharmaceuticalcomposition for treating a disease, disorder, or condition responsive toinhibition of SMYD proteins, such as SMYD3 or SMYD2, or both, whereinthe pharmaceutical composition comprises a therapeutically effectiveamount of a Compound of the Disclosure in a mixture with one or morepharmaceutically acceptable carriers.

In another aspect, the present disclosure provides Compounds of theDisclosure for use in treating cancer in a mammal, e.g., breast,cervical, colon, kidney, liver, head and neck, skin, pancreatic, ovary,esophageal, lung, and prostate cancer.

In another aspect, the present disclosure provides a Compound of theDisclosure for use in the manufacture of a medicament for treatingcancer in a mammal.

In another aspect, the present disclosure provides kit comprising aCompound of the Disclosure.

Additional embodiments and advantages of the disclosure will be setforth, in part, in the description that follows, and will flow from thedescription, or can be learned by practice of the disclosure. Theembodiments and advantages of the disclosure will be realized andattained by means of the elements and combinations particularly pointedout in the appended claims.

It is to be understood that both the foregoing summary and the followingdetailed description are exemplary and explanatory only, and are notrestrictive of the invention as claimed.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present disclosure is based on the use of Compounds ofthe Disclosure as inhibitors of SMYD proteins. In view of this property,the Compounds of the Disclosure are useful for treating diseases,disorders, or conditions, e.g., cancer, responsive to inhibition of SMYDproteins.

One aspect of the present disclosure is based on the use of Compounds ofthe Disclosure as inhibitors of SMYD3. In view of this property, theCompounds of the Disclosure are useful for treating diseases, disorders,or conditions, e.g., cancer, responsive to inhibition of SMYD3.

One aspect of the present disclosure is based on the use of Compounds ofthe Disclosure as inhibitors of SMYD2. In view of this property, theCompounds of the Disclosure are useful for treating diseases, disorders,or conditions, e.g., cancer, responsive to inhibition of SMYD2.

In one embodiment, Compounds of the Disclosure are compounds havingFormula I:

and the pharmaceutically acceptable salts or solvates, e.g., hydrates,thereof, wherein:

R¹ is selected from the group consisting of ethyl and cyclopropyl;R^(2a), R^(2b), R^(3a), and R^(3b) are selected from the groupconsisting of hydrogen and C₁₋₄ alkyl;

X is selected from the group consisting of —N(—Y—Z)— and —CH[N(H)—Y—Z]—;

Y is selected from the group consisting of —C(═O)— and —S(═O)₂—;

Z is selected from the group consisting of —CH═CH₂, —CH—C(H)CH₂NH₂,—CH═C(H)CH₂N(H)CH₃, —CH═C(H)CH₂N(CH₃)₂, —CH═C(H)CH₂CH₂NH₂,—CH═C(H)CH₂CH₂N(H)CH₃, —CH═C(H)CH₂CH₂N(CH₃)₂, —CH₂Br, and —CH₂I;

n is 0 or 1; and

m is 0 or 1.

In another embodiment, Compounds of the Disclosure are compounds havingFormula II:

and the pharmaceutically acceptable salts or solvates, e.g., hydrates,thereof, wherein R¹, R^(2a), R^(2b), R^(3a), R^(3b), Y, m, and n arc asdefined above in connection with Formula I. In another embodiment,R^(2a), R^(2b), R^(3a), and R^(3b) are hydrogen

In another embodiment, Compounds of the Disclosure are compounds havingFormula III:

Formula IV:

Formula V;

or

Formula VI:

and the pharmaceutically acceptable salts or solvates, e.g,, hydrates,thereof, wherein R^(3a) is C₁₋₄ alkyl; and R¹, Y, and Z are as definedabove in connection with Formula I.

In another embodiment, Compounds of the Disclosure are compounds havingFormula VII:

and the pharmaceutically acceptable salts or solvates, e.g., hydrates,thereof, wherein R¹, Y, and Z are as defined above in connection withFormula I.

In another embodiment, Compounds of the Disclosure are compounds havingFormula VIII:

or

Formula IX:

and the pharmaceutically acceptable salts or solvates, e.g., hydrates,thereof, wherein R¹, Y, and Z are as defined above in connection withFormula I.

In another embodiment, Compounds of the Disclosure are compounds havingFormula X:

and the pharmaceutically acceptable salts or solvates, e.g., hydrates,thereof, wherein R^(2a), R^(2b), R^(3a), R^(3b), Y, Z, m, and n are asdefined above in connection with Formula I.

In another embodiment, Compounds of the Disclosure are compounds havingFormula XI:

Formula XII:

and the pharmaceutically acceptable salts or solvates, e.g,, hydrates,thereof, wherein R¹, Y, and Z are as defined above in connection withFormula I.

In another embodiment, Compounds of the Disclosure are compounds havingany one of Formulae I-XII, and the pharmaceutically acceptable salts orsolvates, e.g., hydrates, thereof, wherein Y is —C(═O)—.

In another embodiment, Compounds of the Disclosure are compounds havingany one of Formulae I-XII, and the pharmaceutically acceptable salts orsolvates, e.g., hydrates, thereof, wherein Y is —S(═O)₂—.

In another embodiment, Compounds of the Disclosure are compounds havingany one of Formulae I-XII, and the pharmaceutically acceptable salts orsolvates, e.g., hydrates, thereof, wherein Z is selected from the groupconsisting of —CH═CH₂, —CH═C(H)CH₂NH₂, —CH═C(H)CH₂N(H)CH₃,—CH═C(H)CH₂N(CH₃)₂, —CH═C(H)CH₂CH₂NH₂, —CH═C(H)CH₂CH₂N(H)CH₃,—CH═C(H)CH₂CH₂N(CH₃)₂, and —CH₂O. In another embodiment, Z is selectedfrom the group consisting of —CH═CH₂ and —CH₂Cl.

In another embodiment, Compounds of the Disclosure are compounds havingany one of Formulae I-XII, and the pharmaceutically acceptable salts orsolvates, e.g., hydrates, thereof, wherein R¹ is ethyl.

In another embodiment, Compounds of the Disclosure are compounds havingany one of Formulae I-XII, and the pharmaceutically acceptable salts orsolvates, e.g., hydrates, thereof, wherein R¹ is cyclopropyl.

In another embodiment, Compounds of the Disclosure arc compounds ofTables 1 and 2, and the pharmaceutically acceptable salts or solvates,e.g., hydrates, thereof, or different pharmaceutically acceptable saltthereof.

It should be appreciated that the Compounds of the Disclosure in certainembodiments are the free base, various salts, and hydrate forms, and arenot limited to the particular salt listed in Table 1.

TABLE 1 LCMS SMYD3 SMYD3 Observed Biochem Cell Cpd. Salt M + H IC₅₀ IC₅₀No. Structure Name Form (M + Na) (uM)* (uM)*  1

5-cyclopropyl-N-(1- (vinylsulfonyl)azetidin-3-yl)isoxazole-3-carboxamide HCl 298 >10 >10  2

5-cyclopropyl-N-(1r,4r)-4- (vinylsulfonamido)cyclohexyl)isoxazole-3-carboxamide HCl (362.05) 31.00852  3

N-((1r,4r)-4-acrylamidocyclohexyl)-5- cyclopropylisoxazole-3-carboxamideNone 304.1 74.14102 >10.0  4

N-(1-(2-chloroacetyl)piperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide None 312 33.49521 6.6524  5

N-(1-acryloylpiperidin-4-yl)-5- cyclopropylisoxazole-3-carboxamide None290 >100 >10  6

5-cyclopropyl-N-((2S)-2-methyl-1-(vinylsulfonyl)piperidin-4-yl)isoxazole- 3-carboxamide None 340 5.274762.1188  7

5-cyclopropyl-N-((2R)-2-methyl-1-(vinylsulfonyl)piperidin-4-yl)isoxazole- 3-carboxamide None (362)3.67639 2.19283  8

5-cyclopropyl-N-((1s,4s)-4- (vinylsulfonamido)cyclohexyl)isoxazole-3-carboxamide HCl 340 >10 >10  9

5-cyclopropyl-N-(1- (vinylsulfonyl)piperidin-4-yl)isoxazole-3-carboxamide None (348) 31.65704 10

5-cyclopropyl-N-(1- (vinylsulfonyl)pyrrolidin-3-yl)isoxazole-3-carboxamide None 312 >50 *IC₅₀ values are an average of n = 1 to n =50

TABLE 2 Cpd. No. Structure Name 11

(E)-N-(1-((3-aminoprop-1-en-1- yl)sulfonyl)piperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide 12

(E)-5-cyclopropyl-N-(1-((3- (methylamino)prop-1-en-1-yl)sulfonyl)piperidin-4-yl)isoxazole- 3-carboxamide 13

(E)-5-cyclopropyl-N-(1-((3- (dimethylamino)prop-1-en-1-yl)sulfonyl)piperidin-4-yl)isoxazole- 3-carboxamide 14

(E)-N-(1-((4-aminobut-1-en-1- yl)sulfonyl)piperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide 15

(E)-5-cyclopropyl-N-(1-((4- (methylamino)but-1-en-1-yl)sulfonyl)piperidin-4-yl)isoxazole- 3-carboxamide 16

(E)-5-cyclopropyl-N-(1-((4- (dimethylamino)but-1-en-1-yl)sulfonyl)piperidin-4-yl)isoxazole- 3-carboxamide

Definitions

For the purpose of the present disclosure, the term “alkyl” as used byitself or as part of another group refers to a straight- orbranched-chain aliphatic hydrocarbon containing one to twelve carbonatoms (i.e., C₁₋₁₂ alkyl) or the number of carbon atoms designated(i.e., a C₁ alkyl such as methyl, a C₂ alkyl such as ethyl, a C₃ alkylsuch as propyl or isopropyl, etc.). In one embodiment, the alkyl groupis chosen from a straight chain C₁₋₄ alkyl group. In another embodiment,the alkyl group is chosen from a branched chain C₃₋₄ alkyl group. inanother embodiment, the alkyl group is chosen from a straight orbranched chain C₃₋₄ alkyl group. In another embodiment, the alkyl groupis partially or completely deuterated, i.e., one or more hydrogen atomsof the alkyl group are replaced with deuterium atoms. Non-limitingexemplary C₁₋₄ alkyl groups include methyl, ethyl, propyl, isopropyl,butyl, sec-butyl, tert-butyl, and iso-butyl.

For the purpose of the present disclosure, the term “cycloalkyl” as usedby itself or as part of another group refers to saturated cyclicaliphatic hydrocarbons containing one to three rings having from threeto twelve carbon atoms (i.e,, C₃₋₁₂ cycloalkyl) ar the number of carbonsdesignated. In one embodiment, the cycloalkyl group is cyclopropyl.

The present disclosure encompasses any of the Compounds of theDisclosure being isotopically-labelled (i.e., radiolabeled) by havingone or more atoms replaced by an atom having a different atomic mass ormass number. Examples of isotopes that can be incorporated into thedisclosed compounds include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorous, fluorine and chlorine, such as ²H (or deuterium(D)), ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl,respectively, e.g., ³H, ¹¹C, and ¹⁴C. In one embodiment, provided is acomposition wherein substantially all of the atoms at a position withinthe Compound of the Disclosure are replaced by an atom having adifferent atomic mass or mass number. In another embodiment, provided isa composition wherein a portion of the atoms at a position within theCompound of the disclosure are replaced, i.e., tile Compound of theDisclosure is enriched at a position with an atom having a differentatomic mass or mass number.” Isotopically-labelled Compounds of theDisclosure can be prepared by methods latown in the art.

Compounds of the Disclosure may contain one or more asymmetric centersand may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms. The present disclosure is meant to encompass theuse of all such possible forms, as well as their racemic and resolvedforms and mixtures thereof. The individual enantiomers can be separatedaccording to methods known in the art in view of the present disclosure.When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that they include both E and Z geometric isomers. Alltautomers are intended to be encompassed by the present disclosure aswell.

As used herein, the term “stereoisomers” is a general term for allisomers of individual molecules that differ only in the orientation oftheir atoms in space. It includes enantiomers and isomers of compoundswith more than one chiral center that are not mirror images of oneanother (diastereotners).

The term “chiral center” or “asymmetric carbon atom” refers to a carbonatom to which four different groups are attached,

The terms “enantiomer” and “enantiomeric” refer to a molecule thatcannot be superimposed on its mirror image and hence is optically activewherein the enantionier rotates the plane of polarized light in onedirection and its mirror image compound rotates the plane of polarizedlight in the opposite direction.

The term “racemic” refers to a mixture of equal parts of enantiomers andwhich mixture is optically inactive.

The term “absolute configuration” refers to the spatial arrangement ofthe atoms of a chiral molecular entity (or group) and its stereochemicaldescription, e.g., R or S.

The stereochemical terms and conventions used in the specification aremeant to be consistent with those described in Pure & April. Chem68:2193 (1996), unless otherwise indicated.

The term “enantiomeric excess” or “ee” refers to a measure for how muchof one enantiomer is present compared to the other. For a mixture of Rand S enantiomers, the percent enantiomeric excess is defined as|R—S|*100, where R and S are the respective mole or weight fractions ofenantiomers in a mixture such that R+S=1. With knowledge of the opticalrotation of a chiral substance, the percent enantiomeric excess isdefined as ([α]_(abs)/[α]_(max))*100, where [α]_(abs) is the opticalrotation of the mixture of enantiomers and [α]_(max) is the opticalrotation of tile pare enantiomer. Determination of enantiomeric excessis possible using a variety of analytical techniques, including NMRspectroscopy, chiral column chromatography or optical polarimetry.

The terms “enantiomerically pure” or “enantiopure” refer to a sample ofa chiral substance all of whose molecules (within the limits ofdetection) have the same chirality sense.

The terms “enantiomerically enriched” or “enantioenriched” refer to asample of a chiral substance whose enantiomeric ratio is greater than50:50. Enantiomerically enriched compounds may be enantiomerically pure.

The terms “a” and “an” refer to one or more.

The term “about,” as used herein, includes the recited number ±10%,Thus, “about 10” means 9 to 11.

The present disclosure encompasses the preparation and use of salts ofthe Compounds of the Disclosure, including non-toxic pharmaceuticallyacceptable salts. Examples of pharmaceutically acceptable addition saltsinclude inorganic and organic acid addition salts and basic salts. Thepharmaceutically acceptable salts include, but are not limited to, metalsalts such as sodium salt, potassium salt, cesium salt and the like;alkaline earth metals such as calcium salt, magnesium salt and the like;organic amine salts such as triethylamine salt, pyridine salt, picolinesalt, ethanolamine salt, triethanolamine salt, dicyclohexylarnine salt,N,N′-dibenzylethylenediamine salt and the like; inorganic acid saltssuch as hydrochloride, hydrobromide, phosphate, sulphate and the like;organic acid salts such as citrate, lactate, tartrate, maleate,fumarate, mandelate, acetate, dichloroacetate, triftuoroacetate,oxalate, formate and the like; sulfonates such as methanesulfonate,benzenesulfonate, p-toluenesulfonate and the like; and amino acid saltssuch as arginate, asparginate, glutamate and the like. The term“pharmaceutically acceptable salt” as used herein, refers to any salt,e.g., obtained by reaction with an acid or a base, of a Compound of theDisclosure that: is physiologically tolerated in the target patient(e.g., a mammal, e,g., a human).

Acid addition salts can be formed by mixing a solution of the particularCompound of the Disclosure with a solution of a pharmaceuticallyacceptable non-toxic acid such as hydrochloric acid, fumaric acid,maleic acid, succinic acid, acetic acid, citric acid, tartaric acid,carbonic acid, phosphoric acid, oxalic acid, dichloroacetic acid, or thelike. Basic salts can be formed by mixing a solution of the compound ofthe present disclosure with a solution of a pharmaceutically acceptablenon-toxic base such as sodium hydroxide, potassium hydroxide, cholinehydroxide, sodium carbonate and the like.

The present disclosure encompasses the preparation and use of solvatesof Compounds of the Disclosure. Solvates typically do not significantlyalter the physiological activity or toxicity of the compounds, and assuch may function as pharmacological equivalents. The term “solvate” asused herein is a combination, physical association and/or solvation of acompound of the present disclosure with a. solvent molecule such as,e,g, a disolvate, monosolvate or hemisolvate, where the ratio of solventmolecule to compound of the present disclosure is about 2:1, about 1:1or about 1:2, respectively. This physical association involves varyingdegrees of ionic and covalent bonding, including hydrogen bonding. Incertain instances, the solvate can be isolated, such as when one or moresolvent molecules are incorporated into the crystal lattice of acrystalline solid. Thus, “solvate” encompasses both solution-phase andisolatable solvates. Compounds of the Disclosure can be present assolvated forms with a phartnaceutically acceptable solvent, such aswater, methanol, ethanol, and the like, and it is intended that thedisclosure includes both solvated and unsolvated forms of Compounds ofthe Disclosure. One type of solvate is a hydrate. A “hydrate” relates toa particular subgroup of solvates where the solvent molecule is water.Solvates typically can function as pharmacological equivalents.Preparation of solvates is known in the art. See, for example, M. Cairnet al, Pharmaceut. Sci., 9(3):601-611 (2004), which describes thepreparation of solvates of fluconazole with ethyl acetate and withwater, Similar preparation of solvates, hemisolvates, hydrates, and thelike are described by E. C. van Tonder et al., AAPS Pharm. Sci. Tech.,5(i):Article 12 (2004), and A. L. Bingham et al., Chem. Commun. 603 -604(2001). A typical, non-limiting, process of preparing a solvate wouldinvolve dissolving a Compound of the. Disclosure in a desired solvent(organic, water, or a mixture thereof) at temperatures above 20° C. toabout 25° C., then cooling the solution at a rate sufficient to formcrystals, and isolating the crystals by known methods, e.g., filtration,Analytical techniques such as infrared spectroscopy can be used toconfirm the presence of the solvent in a crystal of the solvate.

Since Compounds of the Disclosure are inhibitors of SMYD proteins, suchas SMYD3 and SMYD2, a number of diseases, conditions, or disordersmediated by SMYD proteins, such as SMYD3 and SMYD2, can be treated byemploying these compounds. The present disclosure is thus directedgenerally to a method for treating a disease, condition, or disorderresponsive to the inhibition of SMYD proteins, such as SMYD3 and SMYD2,in an animal suffering from, or at risk of suffering from, the disorder,the method comprising administering to the animal an effective amount ofone or more Compounds of the Disclosure.

In one aspect, the Compounds of the Disclosure are therapeuticallyeffective inhibitors of SMYD proteins by irreversibly binding one ormore SMYD proteins such as SMYD3 or SMYD2. In some embodiments, theCompounds of the Disclosure are therapeutically effective inhibitors ofSMYD proteins by forming covalent bonds with one or more SMYD proteinssuch as SMYD3 or SMYD2.

The present disclosure is further directed to a method of inhibitingSMYD proteins in an animal in need thereof, the method comprisingadministering to the animal a therapeutically effective amount of atleast one Compound of the Disclosure.

The present disclosure is further directed to a method of inhibitingSMYD3 in an animal in need thereof, the method comprising administeringto the animal a therapeutically effective amount of at least oneCompound of the Disclosure,

The present disclosure is further directed to a method of inhibitingSMYD2 in an animal in need thereof, the method comprising administeringto the animal a therapeutically effective amount of at least oneCompound of the Disclosure.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to eliminating, reducing, or atneliorating a disease or condition,andlor symptoms associated therewith. Although not precluded, treating adisease or condition does not require that the disease, condition, orsymptoms associated therewith be completely eliminated. As used herein,the terms “treat,” “treating,” “treatment,” and the like may include“prophylactic treatment,” which refers to reducing the probability ofredeveloping a disease or condition, or of a recurrence of apreviously-controlled disease or condition, in a subject who does nothave, but is at risk of or is susceptible to, redeveloping a disease orcondition or a recurrence of the disease or condition. The term “treat”and synonyms contemplate administering a therapeutically effectiveamount of a Compound of the Disclosure to an individual in need of suchtreatment.

Within the meaning of the disclosure, “treatment” also includes relapseprophylaxis or phase prophylaxis, as web as the treatment of acute orchronic signs, symptoms and/or malfunctions. The treatment can heorientated symptomatically, for example, to suppress symptoms. It can beeffected over a short period, be oriented over a medium term, or can bea long-term treatment, for example within the context of a maintenancetherapy.

The term “therapeutically effective amount” or “effective dose” as usedherein refers to an amount of the active ingredient(s) that is(are)sufficient, when administered by a method of the disclosure, toefficaciously deliver the active ingredient(s) for the treatment ofcondition or disease of interest to an individual in need thereof. Inthe case of a cancer or other proliferation disorder, thetherapeutically effective amount of the agent may reduce (i.e., retardto some extent and preferably stop) unwanted cellular proliferation;reduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,retard to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., retard to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; modulateprotein methylation in the target cells; and/or relieve, to some extent,one or more of the symptoms associated with the cancer. To the extentthe administered compound or composition prevents growth and/or killsexisting cancer cells, it may be cytostatic and/or cytotoxic.

The term “container” means any receptacle and closure therefore suitablefor storing, shipping, dispensing, and/or handling a pharmaceuticalproduct.

The term “insert” means information accompanying a pharmaceuticalproduct that provides a description of how to administer the product,along with the safety and efficacy data required to allow the physician,pharmacist, and patient to make an informed decision regarding use ofthe product. The package insert generally is regarded as the “label” fora pharmaceutical product.

The term “disease” or “condition” or “disorder” denotes disturbancesand/or anomalies that as a rule are regarded as being pathologicalconditions or functions, and that can manifest themselves in the form ofparticular signs, symptoms, and/or malfunctions. As demonstrated below,Compounds of the Disclosure inhibit SMYD proteins, such as SMYD3 andSMYD2 and can be used in treating diseases and conditions such asproliferative diseases, wherein inhibition of SMYD proteins, such asSMYD3 and SMYD2 provides a benefit.

In some embodiments, the Compounds of the Disclosure can be used totreat a “SMYD protein mediated disorder” (e.g., a SMYD3-mediateddisorder or a SMYD2-mediated disorder). A SMYD protein mediated disorderis any pathological condition in which a SMYD protein is know to play arole. In some embodiments, a SMYD-mediated disorder is a proliferativedisease.

In some embodiments inhibiting SMYD proteins, such as SMYD3 and SMYD2,is the inhibition of the activity of one or more activities of SMYDproteins such as SMYD3 and SMYD2. In some embodiments, the activity ofthe SMYD proteins such as SMYD3 and. SMYD2 is the ability of tile SMYDprotein such as SMY3 or SMYD2 to transfer a methylgroup to a targetprotein histone). It should be appreciated that the activity of the oneor more SivIYD proteins such as SMYD3 and SMYD2 may be inhibited in vivoor in vivo. Examplary levels of inhibition of the activity one or moreSMYD proteins such as SMYD3 and SMYD2 include at least 10% inhibiton, atleast 20% inhibition, at least 30% inhibitor, at least 40% inhibition,at least 50% inhibitor, at least 60% inhibition, at least 70% inhibiton,at least 80% inhibition, at least 90% inhibiton, and up to 100%inhibition.

The SMYD (SET and MYND domain) family of lysine methyltransferases(KMTs) plays pivotal roles in various cellular processes, including geneexpression regulation and DNA damage response. The family of human SATYDproteins consists of SMYD1, SMYD2, SMYD3, SMYD4 and SMYD5. SMYD1, SMYD2,and SMYD3 share a high degree of sequence homology and, with theexception of SMYD5, human SMYD proteins harbor at least one C-terminaltetratrieo peptide repeat (TPR) domain. (See e.g., Abu-Farha et at. JMol Cell Biol (2011) 3 (5) 301-308). The SMYD proteins have been foundto be linked to various cancers (See e.g., Hamamoto et al.Nat Cell.Biol, 2004, 6: 731-740), Hu et al. Canncer Research 2009, 4067-4072, andKomatsu et al. Carcinogenesis 2009, 301139-1146.)

SMYD3 is a protein methyltransfera.se found to be expressed at highlevels in a. number of different cancers (Hamamoto, R., et al., Nat.Cell Biol., 6(8):731-40 (2004)). SMYD3 likely plays a role in theregulation of gene transcription and signal transduction pathwayscritical for survival of breast, liver, prostate and lung cancer celllines (Hamamoto, R., et al., Nat. Cell Biol., 6(8):731-40 (2004);Hamamoto, R., et al., Cancer Sci., 97(2):113-8 (2006); Van Alter, G. S.,et al., Epigenetics, 7(4):340-3 (2012); Liu, C., et al., J. Natl. CancerInst., 105(22):1719-28 (2013); Mazur, P. K., et al., Nature,510(7504):283-7 (2014)).

Genetic knockdown of SMYD3 leads to a decrease in proliferation of avariety of cancer cell lines (Hamamoto, R., et al., Nat. Cell Biol.,6(8):731-40 (2004); Hamamoto, R., et al., Cancer Sci., 97(2):113-8(2006); Van Alter, G. S., et al., Epigenetics, 7(4):340-3 (2012); Liu,C., et al J. Natl. Cancer Inst., 105(22):1719-28 (2013); Mazur, P. K.,et al., Nature, 510(7504):283-7 (2014)). Several studies employingRNAi-based technologies have shown that ablation of SMYD3 inhepatocellular carcinoma cell lines greatly reduces cell viability andthat its pro-survival role is dependent on its catalytic activity(Hamamoto, R., et al., Nat. Cell Biol., 6(8):731-40 (2004); Van Aller,G. S., et al., Epigenetics, 7(4):340-3 (2012)). Moreover, SMYD3 has alsobeen shown to be a critical mediator of transformation resulting fromgain of function mutations in the oncogene, KRAS for both pancreatic andlung adenocarcinoma in mouse models. The dependence of KRAS on SMYD3 wasalso shown to be dependent on its catalytic activity (Mazur, P. K., etal., Nature, 510(7504):283-7 (2014)).

SMYD2 (SET and MYND domain-containing protein 2) was first characterizedas protein that is a member of a sub-family of SET domain containingproteins which catalyze the site-specific transfer of methyl groups ontosubstrate proteins. SMYD2 was initially shown to have methyltransferaseactivity towards lysine 36 on histone H3 (H3K36) but has subsequentlybeen shown to have bath histone and non-histone methyltrasferaseactivity.

SMYD2 has been implicated in the pathogenesis of multiple cancers. Ithas been shown to be over-expressed, compared to matched normal samples,in tumors of the breast, cervix, colon, kidney, liver, head and neck,skin, pancreas, ovary, esophagus and prostate, as well as hematologicmalignancies such as AML, B- and T-ALL, CLL, and MCL, suggesting a rolefor SMYD2 in the biology of these cancers. More specifically, studiesusing genetic knock-down of SMYD2 have demonstrated anti-proliferativeeffects in esophageal squamous cell carcinoma (ESCC), bladder carcinomaand cervical carcinoma cell lines. Moreover, high expression of SMYD2has been shown to be a poor prognostic factor in both ESCC and pediatricALL.

In one aspect, the present disclosure provides a method of treatingcancer in a. patient comprising administering a therapeuticallyeffective amount of a Compound of the Disclosure. While not beinglimited to a specific mechanism, in some embodiernms, Compounds of theDisclorure can treat cancer by inhibiting SMYD proteins, such as SMYD3and SMYD2. Examples of treatable cancers include, but are not limitedto, adrenal cancer, acinic cell carcinoma, acoustic neuroma, acrallentigious melanorna, acrospirorna, acute eosinophilic leukemia, acuteerythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblasticleukemia, acute monocytic leukemia, acute promyelocytic leukemia,adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoidodontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm,adrenocortical carcinoma, adult T-cell leukemiallymphoma, aggressiveNK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma,alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large celllymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma,angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoidtumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocyticleukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer,bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma insitu, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma,chondroma, chordoma, choriocarcinoma, choroid plexus papilloma,clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-celllymphoma, cervical cancer, colorectal cancer, Degos disease,desmoplastic small round cell tumor, diffuse large B-cell lymphoma,dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonalcarcinoma, endocrine gland neoplasm, endodermal sinus tumor,enteropathy-associated T-cell lymphoma, esophageal cancer, fetus infetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroidcancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor,gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumorof the hone, glial tumor, glioblastoma multiforme, glioma, gliomatosiscerebri, glucagonoma, gonadoblastoma, granulosa cell tumor,gynandroblastoma, gallbladder cancer, gastric cancer, hairy cellleukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma,hematological malignancy, hepatoblastoma, hepatosplenic lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma,intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna,lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lungcancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma,acute lymphocytic leukemia, acute myelogeous leukemia, chroniclymphocytic leukemia, liver cancer, small cell lung cancer, non-smallcell lung cancer, MALT lymphoma, malignant fibrous histiocytoma,malignant peripheral nerve sheath tumor, malignant triton tumor, mantlecell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia,mediastinal germ cell tumor, medullary carcinoma of the breast,medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merketcell cancer, mesothelioma, metastatic urothelial carcinoma, mixedMullerian tumor, mucinous tumor, multiple myeloma, muscle tissueneoplasm, mycosis fungoidcs, myxoid liposarcoma, myxoma, myxosarcoma,nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma,neuroma, nodular melanoma, ocular cancer, oligoastrocytoma,oligodendroglioma, oncocytoma, optic nerve sheath meningioma, opticnerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor,papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma,pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma,polyembryoma, precursor T-lymphoblastic lymphoma, primary centralnervous system lymphoma, primary effusion lymphoma, preimary peritonealcancer, prostate cancer, pancreatic cancer, pharyngeal cancer,pseudomyxoma periotonei, renalcell carcinoma, renal medullary carcinoma,retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation,rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor,sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer,small blue round cell tumors, small cell carcinoma, soft tissue sarcoma,somatostatinoma, soot wart, spinal tumor, splenic marginal zonelymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease,small intestine cancer, squamous carcinoma, stomach cancer, T-celllymphoma, testicular cancer, thecoma, thyroid cancer, transitional cellcarcinoma, throat cancer, urachal cancer, urogenital cancer, urothelialcarcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visualpathway vulva r cancer, vaginal cancer, Waldenstrom'sm.acroglobulinernia, Warthin's tumor, and Wilms' tumor.

In another embodiment, the cancer is breast, cervix, colon, kidney,liver, head and neck, skin, pancreas, ovary, esophagus, or prostatecancer.

In another embodiment, the cancer is a hematologic malignancy such asacute myeloid leukemia (AML), B- and T-acute lymphoblastic leukemia(ALL), chronic lymphocytic leukemia (CLL), or mantle cell lymphoma(MCL).

In another embodiment, the cancer is esophageal squamous cell carcinoma(ESCC), bladder carcinoma, or cervical carcinoma.

In another embodiment, the cancer is a leukemia, for example a leukemiaselected from acute monocytic leukemia, acute myelogenous leukemia,chronic myelogenous leukemia, chronic lymphocytic leukemia and mixedlineage leukemia (MLL). In another embodiment the cancer is NUT-midlinecarcinoma. In another embodiment the cancer is multiple myeloma. Inanother embodiment the cancer is a lung cancer such as small cell lungcancer (SCLC). In another embodiment the cancer is a neuroblastoma. Inanother embodiment the cancer is Burkitt's lymphoma. In anotherembodiment the cancer is cervical cancer. In another embodiment thecancer is esophageal cancer. In another embodiment the cancer is ovariancancer. In another embodiment the cancer is colorectal cancer. lnanother embodiment, the cancer is prostate cancer. In anotherembodiment, the cancer is breast cancer.

In another embodiment, the present disclosure provides a therapeuticmethod of modulating protein methylation, gene expression, cellproliferation, cell differentiation and/or apoptosis in vivo in thecancers mentioned above by administering a therapeutically effectiveamount of a Compound of the Disclosure to a subject in need of suchtherapy.

Compounds of the Disclosure can be administered to a mammal in the formof a raw chemical without any other components present. Compounds of theDisclosure can also be administered to a mammal as part of apharmaceutical composition containing the compound combined with asuitable pharmaceutically acceptable carrier. Such a carrier can beselected from pharmaceutically acceptable excipients and auxiliaries Theterm “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable vehicle” encompasses any of the standard pharmaceuticalcarriers, solvents, surfactants, or vehicles. Suitable pharmaceuticallyacceptable vehicles include aqueous vehicles and nonaqueous vehicles.Standard pharmaceutical carriers and their formulations are described inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,19th ed. 1995.

Pharmaceutical compositions within the scope of the present disclosureinclude all compositions where a Compound of the Disclosure is combinedwith one or more pharmaceutically acceptable carriers. In oneembodiment, the Compound of the Disclosure is present in the compositionin an amount that is effective to achieve its intended therapeuticpurpose. While individual needs may vary, a determination of optimalranges of effective amounts of each compound is within the skill of theart, Typically, a Compound of the Disclosure can be administered to amammal, e.g., a human., orally at a dose of from about 0.0025 to about1500 mg per kg body weight of the mammal, or an equivalent amount of apharmaceutically acceptable salt or solvate thereof, per day to treatthe particular disorder. A useful oral dose of a Compound of theDisclosure administered to a mammal is from about 0.0025 to about 50 mgper kg body weight of the mammal, or an equivalent amount of thepharmaceutically acceptable salt or solvate thereof. For intramuscularinjection, the dose is typically about one-half of the oral dose.

A unit oral dose may comprise from about 0.01 mg to about 1 g of theCompound of the Disclosure, about 0.01 mg to about 500 mg, about 0.01 mgto about 250 mg, about 0.01 mg to about 100 mg, 0.01 mg to about 50 mg,e.g., about 0.1 mg to about 10 mg, of the compound. The unit dose can beadministered one or more times daily, as one or more tablets orcapsules, each containing from about 0.01 mg to about 1 g of thecompound, or an equivalent amount of a pharmaceutically acceptable saltor solvate thereof.

A pharmaceutical composition of the present disclosure can beadministered to any patient that may experience the beneficial effectsof a Compound of the Disclosure. Foremost among such patients aremammals, e.g., humans and companion animals, although the disclosure isnot intended to be so limited. In one embodiment, the patient is ahuman.

A pharmaceutical composition of the present disclosure can beadministered by any means that achieves its intended purpose. Forexample, administration can be by the oral, parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, intranasal,transmucosal, rectal, intravagiaal or buccal route, or by inhalation.The dosage administered and route of administration will vary, dependingupon the circumstances of the particular subject, and taking intoaccount such factors as age, gender, health, and weight of therecipient, condition or disorder to he treated, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired.

In one embodiment, a pharmaceutical composition of the presentdisclosure can be administered orally. in another embodiment, apharmaceutical composition of the present disclosure can be administeredorally and is formulated into tablets, dragees, capsules, or an oralliquid preparation. In one embodiment, the oral formulation comprisesextruded multipartieulates comprising the Compound of the Disclosure.

Alternatively, a pharmaceutical composition of the present disclosurecan be administered rectally, and is formulated in suppositories.

Alternatively, a pharmaceutical composition of the present disclosurecan be administered by injection.

Alternatively, a pharmaceutical position of the present disclosure canbe administered transdermally.

Alternatively, a pharmaceutical composition of the present disclosurecan be administered by inhalation or by intranasal or transmucosaladministration.

Alternatively, a pharmaceutical composition of the present disclosurecan be administered by the intravaginal route.

A pharmaceutical composition of the present disclosure can contain fromabout 0.01 to 99 percent by weight, e.g., from about 0.25 to 75 percentby weight, of a Compound of the Disclosure.

A pharmaceutical composition of the present disclosure is manufacturedin a manner which itself will be known in view of the instantdisclosure, for example, by means of conventional mixing, granulating,dragee-making, dissolving, extrusion, or lyophilizing processes. Thus,pharmaceutical compositions for oral use can be obtained by combiningthe active compound with solid excipients, optionally grinding theresulting mixture and processing the mixture of granules, after addingsuitable auxiliaries, if desired or necessary, to obtain tablets ordragee cores.

Suitable excipients include fillers such as saccharides (for example,lactose, sucrose, mannitol or sorbitol), cellulose preparations, calciumphosphates (for example, tricalcium phosphate or calcium hydrogenphosphate), as well as binders such as starch paste (using, for example,maize starch, wheat starch, rice starch, or potato starch), gelatin,tragacanth, methyl cellulose, hydrowropyltnethylcollulose, sod iumcarboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, one ormore disintegrating agents can be added, such as the above-mentionedstarches and also carboxymethyl-starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodiumalginate.

Auxiliaries are typically flow-regulating agents and lubricants such as,for example, silica, talc, stearic acid or salts thereof (e,g,,magnesium stearate or calcium stearate), and polyethylene glycol. Drageecores are provided with suitable coatings that are resistant to gastricjuices. For this purpose, concentrated saccharide solutions can be used,which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropytmethyl-cellulose phthalate can be used. Dye stuffs orpigments can be added to the tablets or dragee coatings, for example,for identification or in order to characterize combinations of activecompound doses.

Examples of other pharmaceutical preparations that can be used orallyinclude push-fit capsules made of gelatin, or soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain a compound in the form of granules, which can bemixed with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers, or in the form of extruded multiparticulates. In softcapsules, the active compounds are preferably dissolved or suspended insuitable liquids, such as fatty oils or liquid paraffin. In addition,stabilizers can be added.

Possible pharmaceutical preparations for rectal administration include,for example, suppositories, which consist of a combination of one ormore active compounds with a suppository base. Suitable suppositorybases include natural arid synthetic triglycerides, and paraffinhydrocarbons, among others. It is also possible to use gelatin rectalcapsules consisting of a combination of active compound with a basematerial such as, for example, a liquid triglyceride, polyethyleneglycol, or paraffin hydrocarbon.

Suitable formulations for parenteral administration include aqueoussolutions of the active compound in a water-soluble form such as, forexample, a water-soluble salt, alkaline solution, or acidic solution.Alternatively, a suspension of the active compound can be prepared as anoily suspension. Suitable lipophilic solvents or vehicles for such assuspension may include fatty oils (for example, sesame oil), syntheticfatty acid esters (for example, ethyl oleate), triglycerides, or apolyethylene glycol such as polyethylene glycol-400 (PEG-400). Anaqueous suspension may contain one or more substances to increase theviscosity of the suspension, including, for example, sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. The suspension mayoptionally contain stabilizers.

In another embodiment, the present disclosure provides kits whichcomprise a Compound of the Disclosure (or a composition comprising aCompound of the Disclosure) packaged in a manner that facilitates theiruse to practice methods of the present disclosure. In one embodiment,the kit includes a Compound of the Disclosure (or a compositioncomprising a Compound of the Disclosure) packaged in a container, suchas a sealed bottle or vessel, with a label affixed to the container orincluded in the kit that describes use of the compound or composition topractice the method of the disclosure. In one embodiment, the compoundor composition is packaged in a unit dosage form. The kit further caninclude a device suitable for administering the composition according tothe intended route of administration.

General Synthesis of Compounds

Compounds of the Disclosure are prepared using methods known to thoseskilled in the art in view of this disclosure, or by the illustrativemethods shown in the General Schemes below. In the General Schemes, R¹,R^(2a), R^(2b), R^(3a), R^(3b), n, and Z of Formulae A-C are as definedin connection with Formula I, unless otherwise indicated. In any of theGeneral Schemes, suitable protecting can be employed in the synthesis,for example, when Z is CH═C(H)CH₂NH₂, or any other group that may requieprotection. (See, Wuts, P. G. M.; Greene, T. W., “Greene's ProtectiveGroups in Organic Synthesis”, 4th Ed., J. Wiley & Sons, NY, 2007).

Compound A is converted to compound B (i.e, a. compound having FormulaI, wherein Y is —S(═O)₂—) by coupling with a suitable sulfonyl chloride(Z—SO₂Cl) in the presence of a suitable base such as TEA or DIPEA in asuitable solvent such as dichloromethane, acetonitrile, or DMF,

Compound A is converted to compound C (i.e, a compound having Formula I,wherein Y is —C(═O)—) by coupling with a suitable aside chloride(Z—COCl) in the presence of a suitable base such as TEA or DIPEA in asuitable solvent such as dichloromethane, acetonitrile, or DMF, or bycoupling with a suitable carboxylic acid (Z—CO₂H) in the presence of asuitable coupling reagent such as HATU and a suitable base such as TEAor DIPEA in a suitable solvent such as dichloromethane, acetonitrile, orDMF.

EXAMPLES General Synthetic Methods

General methods and experimental procedures for preparing andcharacterizing compounds of Table 1 are set forth in the general schemesabove and the examples below. Wherever needed, reactions were heatedusing conventional hotplate apparatus or heating mantle or microwaveirradiation equipment. Reactions were conducted with or withoutstirring, under atmospheric or elevated pressure in either open orclosed vessels. Reaction progress was monitored using conventionaltechniques such as TLC, HPLC, UPLC, or LCMS using instrumentation andmethods described below. Reactions were quenched and crude compoundsisolated using conventional methods as described in the specificexamples provided. Solvent removal was carried out with or withoutheating, under atmospheric or reduced pressure, using either a rotary orcentrifugal evaporator.

Compound purification was carried out as needed using a variety oftraditional methods including, but not limited to, preparativechromatography under acidic, neutral, or basic conditions using eithernormal phase or reverse phase HPLC or flash columns or Prep-TLC plates.Compound purity and mass confirmations were conducted using standardHPLC and/or UPLC and/or MS spectrometers and/or LCMS and/or GC equipment(i.e., including, but not limited to the following instrumentation:Waters Alliance 2695 with 2996 PDA detector connected with ZQ detectorand ESI source; Shimadzu LDMS-2020; Waters Acquity H Class with PDAdetector connected with SQ detector and ESI source; Agilent 1100 Serieswith PDA detector; Waters Alliance 2695 with 2998 PDA detector; AB SCIEXAPI 2000 with ESI source; Agilent 7890 GC). Exemplified compounds weredissolved in either MeOH or MeCN to a concentration of approximately 1mg/mL and analyzed by injection of 0.5-10 μL into an appropriate LCMSsystem using the methods provided in the following table:

Flow Rate MS Heat Block MS Detector Method Column Mobile Phase A MobilePhase B (mL/min) Gradient Profile Temp (° C.) Voltage (kV) A Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 2.0 minutes, 250 1.5 XR-ODS TFATFA 100% B for 1.1 minutes. 2.2 μm 100% to 5% B in 0.2 minutes, 3.0 × 50mm then stop B Gemini-NX Water/0.04% ACN 1 5% to 100% B in 2.0 minutes.200 0.75 3 μm C18 Ammonia 100% B for 1.1 minutes, 110 A 100% to 5% B in0.1 minutes, then stop C Shim-pack Water/0.05% ACN/0.05% 1 5% to 100% Bin 2.0 minutes, 250 0.85 XR-ODS TFA TFA 100% B for 1.1 minutes, 1.6 μm100% to 5% B in 0.1 minutes, 2.0 × 50 mm then stop D Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 2.0 minutes, 250 0.95 XR-ODS TFATFA 100% B for 1.1 minutes, 2.2 μm 100% to 5% B in 0.1 minutes, 3.0 × 50mm then stop

Compound structure confirmations were carried out using standard 300 or400 MHz NMR spectrometers with nOe's conducted whenever necessary.

The following abbreviations may be used herein:

Abbreviation Meaning ACN acetonitrile atm. atmosphere DCMdichloromethane DHP dihydropyran DIBAL diisobutyl aluminum hydride DIEAdiisopropyl ethylamine DMF dimethyl formamide DMF-DMA dimethyl formamidedimethyl acetal DMSO dimethyl sulfoxide Dppf1,1′-bis(diphenylphosphino)ferrocene EA ethyl acetate ESI electrosprayionization EtOH Ethanol FA formic acid GC gas chromatography H hour Hexhexanes HMDS hexamethyl disilazide HPLC high performance liquidchromatography IPA Isopropanol LCMS liquid chromatography/massspectrometry MeOH Methanol Min Minutes NBS N-bromo succinimide NCSN-chloro succinimide NIS N-iodo succinimide NMR nuclear magneticresonance nOe nuclear Overhauser effect Prep. Preparative PTSApara-toluene sulfonic acid Rf retardation factor rt room temperature RTretention time sat. Saturated SGC silica gel chromatography TBAFtetrabutyl ammonium fluoride TEA Triethylamine TFA trifluoroacetic acidTHF Tetrahydrofuran TLC thin layer chromatography UPLC ultra performanceliquid chromatography

Example 1 Synthesis of 5-cyclopropylisoxazoie-3-carboxylic acid

Step 1: Synthesis of ethyl 4-cyclopropyl-2,4-dioxobutanoate

Into a 10-L 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen Na (164 g, 1.20 equiv) was added inportions to ethanol (5 L). A solution of (CO₂Et)₂ (869 g, 1.00 equiv)and 1-cyclopropylethan-1-one (500 g, 5.94 mol, 1.00 equiv) was addeddropwise with stirring at 0-20° C. The resulting solution was stirredfor 1 h at 20-30° C. and then for an additional 1 h at 80° C. Theresulting solution was diluted with 15 L of H₂O, The pH was adjusted to2 with hydrochloric acid (12N), The resulting solution was extractedwith ethyl acetate and the organic layers combined and washed, withNaHCO₃ (sat. aq.). The extract was concentrated under vacuum yielding820 g (crude) of ethyl 4-cyclopropyl-2,4-dioxobutanoate as yellow oil.TLC (ethyl acetate/petroleum ether=1/5): Rf=0.5.

Step 2: Synthesis of ethyl 5-cyclopropylisoxazole-3-carboxylate

Into a 10 L round-bottom flask, was placed a solution of ethyl4-cyclopropyl-2,4-dioxobutanoate (177 g) in ethanol (1.1 L) andNH₂OH—HCl (200 g). The resulting solution was stirred for 1 h at 20-80°C. The resulting solution was allowed to react, with stirring, for anadditional 1 h at 80° C. The resulting mixture was concentrated undervacuum. The residue was purified on a silica gel column with ethylacetate/petroleum ether (1/10). This resulted in 143 g (the two stepyield was 66.3%) of ethyl 5-cyclopropylisoxazole-3-carboxylate as ayellow oil. TLC (ethyl acetate/petroleum ether=1/5): Rf=0.2.

Step 3: Synthesis of 5-cyclopropylisoxazole-3-carboxylic acid

Into a 10-L round:bottom flask was placed ethyl5-cyclopropylisoxazole-3-carboxylate (280 g, 1.55 mol, 1.00 equiv) and asolution of sodium hydroxide (74.3 g, 1.20 equiv) in water (4 L). Theresulting solution was stirred for 1 h at room temperature, Theresulting mixture was washed with ether. The pH value of the aqueoussolution was adjusted to 2-3 with hydrochloric acid (12N). The resultingsolution was extracted with ethyl acetate and the organic layerscombined and concentrated under vacuum. This resulted in 220 g (93%) of5-cyclopropylisoxazole-3-carboxylic acid as an off-white solid. LCMS(method A, ESI): RT=1.99 min, m/z 153.9 [M+H]⁺. (300 MHz CDCl₃):8.42(brs, 1H), 6.37(s, 1H), 2.16-2.05(m, 1.29-1.12(m, 2H), 1.12-0.99(m,2H) ppm.

Example 2 Synthesis of N-((1r,4 -4-aminocyclohexyl)-5-cyclopropylisoxazole-3-carboxamide hydrochloride

Step 1: Synthesis of tort-butyl(1r,4r)-4-(5-cyclopropylisoxazole-3-carboxamido)cyclohexylcarbamate

In a 100-mL round-bottom flask 5-cyclopropylisoxazole-3-carboxylic acid(100 mg, 0.65 nimol, 1,00 equiv), fert:butylN-[(1r,4r)-4-aminocyclohexyl]carbamate (154 mg, 0.72 mmol, 1.10 equiv)and TEA (198 mg, 1.96 mmol, 3.00 equiv) were dissolved in 10 mldichloromethane, then HATU (496 mg, 1.31 mmol, 2.00 equiv) was added tothe solution. The resulting solution was stirred overnight at roomtemperature. The mixture was then concentrated under vacuum. The residuewas purified on a silica gel column with ethyl acetate/petroleum ether(4:1). This resulted in 210 mg (92%) tert-butyl(1r,4r)-4-(5-cyclopropylisoxazole-3-carboxamido)cyclohexylcarbamate as awhite solid. LCMS (method A, ESI): RT=1.48 min, m/z=294.0 [M-56]⁺.

Step 2: Synthesis ofN-((1r,1r)-4-aminocyclohexyl)-5-cyclopropylisoxazole-3-carboxamidehydrochloride

Into a 250-mL round-bottom flask was placed tert-butyl(1r,4r)-4-(5-cyclopropylisoxazole-3-carboxamido)cyclohexylcarbamate (210mg, 0.60 mmol, 1.00 equiv) and 1,4-dioxane (20 mL). This was followed bythe addition of hydrogen chloride (2M in dioxane, 20 mL). The resultingsolution was stirred overnight at room temperature. The solids werecollected by filtration. This resulted in 140 mg (93%) ofN-((1r,4r)-4-aminocyclohexyl)-5-cyclopropylisoxazole-3-carboxamidehydrochloride as a white solid. ¹H-NMR (300 MHz, D₂O): δ 6.62 (s, 1H),3.82-3.69 (m, 1H), 3.21-3.17 (m, 1H), 2.13-1.92 (m, 5H), 1.57-1.33 (m,4H), 1.10-1.00 (m, 2H), 0.93-0.84 (m, 2H) ppm. LCMS (method D, ESI):RT=0.99 min, m/z=291.0[M+41]⁺.

Example 3 Synthesis of N-((1r,4r)-4-acrylamidocyciohexyl)-5-cyclopropylisoxazole-3-carboxamide

Into a 50-mL round-bottom flask was placed prop-2-enoic acid (108 mg,1.50 mmol, 1.50 equiv),N-((1r,4r)-4-aminocyclohexyl)-5-cyclopropylisoxazole-3-carboxamidehydrochloride (285 mg, 1.00 mmol, 1.00 equiv), dichloromethane (10 mL),TEA (300 mg, 2.97 mmol, 2.98 equiv), and HATU (760 mg, 2.00 mmol, 2.01equiv). The resulting solution was stirred for 6 h at room temperature.The mixture was concentrated under vacuum and the residue purified on aC18 gel column with CH₃CN/H₂O (3:2). This resulted in 57.5 mg (19%) ofN-((1r,4r)-4-acrylamidocyclohexyl)-5-cyclopropylisoxazole-3-carboxamideas a white solid. ¹H-NMR (300 MHz, DMSO-d6): δ 8.49 (d, J=8.1 Hz, 1H),8.00 (d, J=7.5 Hz, 1H), 6.47 (s, 1H), 6.28-6.00 (m, 2H), 5.63-5.50 (m,1H), 3.81-3.64 (m, 1H), 3.64-3.46 (m, 2.25-2.12 (m, 1H), 1.91-1.70 (m,4H), 1.65-1.15 (m, 4H), 1.13-4.02 (m, 2H), 0.98-0.85 (m, 2H) ppm. LCMS(method D, ESI): RT=1.55 min, m/z=304.1 [M+H].

Example 4 Synthesis of5-cyclopropyl-N-((1r,4r)-4-(vinylsulfonamido)cyclohexyl)isoxazole-3-carboxamide(Cpd. No. 2)

Into a 250-mL round-bottom flask was placedN-((1r,4r)-4-aminocyclohexyl)-5-cyclopropylisoxazole-3-carboxamidehydrochloride (300 mg, 1.20 mmol, 1.00 equiv). This was followed by theaddition of dichloromethane (40 mL) and TEA (316 mg, 3.13 mmol, 3.00equiv). Then ethenesulfonyl chloride (263 mg, 2.08 mmol, 2.00 equiv) wasadded dropwise over 5 minutes at room temperature. The resultingsolution was stirred at room temperature for 48 hours. The resultingmixture was concentrated under vacuum and the residue purified on asilica get column with ethyl acetate/petroleum ether (13:7). The productwas further purified by Flash-Prep-HPLC with the following conditions(Prep-HPLC-025): Column, XRridge Prep C18 ORD Column,5 um,19×150 mm,mobile phase, WATER WITH 0.05% TFA and MeCN (5.0% MeCN up to 21.0% in 10min); Detector, LTV 254/220 nm. This resulted in 25.1 mg (6%) of5-cyclopropyl-N-(1r,4r)-4-(vinylsulfonamido)cyclohcxyl)isoxazole-3-carboxamideas a white solid. ¹H-NMR (300 MHz, MeOD): δ 6.73-6.44 (m, 1H), 6.35 (s,1H), 6.14 (d, J=9.1 Hz, 1H), 5.92 (d, J=7.5 Hz, 1H), 3.90-3.75(m, 1H),3.18-3.02(m, 1H), 2.20-2.09(m, 1H), 2.09-1.90(m, 4H), 1.55-1.34 (m, 4H),1.08-1.18 (m, 2H), 1.01-0.91(m, 2H). LCMS (method A, ESI): RT=1.77 min,m/z=362.1 [M+Na]⁺.

Example 5 Synthesis ofN-(1-acryloyipiperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide (Cpd.No. 5)

Into a 50-ml, round-bottom flask was placed5-cyclopropyl-N-(piperidin-4-yl)isoxazole-3-carboxamide hydrochloride(270 mg, 0.99 mmol, 1.00 equiv), prop-2-enoic acid (86 mg, 1.19 mmol,1.20 equiv), dichloromethane (15 mL), HATU (760 mg, 2.00 mmol, 2.01equiv). Then TEA (300 mg, 2.96 mmol, 2.98 equiv) was added dropwise. Theresulting solution was stirred for 15 h at room temperature. The mixturewas concentrated under vacuum and the residue purified on a C18 gelcolumn with CH₃CN/H₂O (1:1). This resulted in 58.9 mg (20%) ofN-(1-acryloylpiperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide as awhite solid. ¹H-NMR (300 MHz, DMSO-d6): δ 8.61 (d, J=8.1 Hz, 1H), 6.83(dd, J=15.8 and 10.5, 1H), 6.49 (s, 1H), 6.09 (dd, J=15.8 and 2.4 Hz,1H), 5.67 (dd, J=10.5 and 2.4 Hz, 1H), 4.50-428 (m, 1H), 4.15-3.90 (m,2H), 3.24-3,05 (m, 1H), 2.85-2.65 (m, 1H), 2.25-2.11 (m, 1H), 1.88-1.71(m, 2H), 1.55-1.30 (m, 2H), 1.06-1.01 (m, 2H), 0.97-0.85 (m, 2H) ppm.LCMS (method D, ESI): RT=1.50 min, m/z=290.0 [M+H]⁺

Example 6 Synthesis of 5-cyclpropyl-N-(1-(vinvisulfonyl)azetidin-3-yt)isoxazoie-3-carboxamide (Cpd.No. 1)

Step 1: Synthesis of tert-butyl3-(5-cyclopropylisoxazole-3-carboxamido)azetidine-1-carboxylate

Into a 100-mL round-bottom flask, was placed tert-butyl3-aminoazetidine-1 -carboxylate (500 mg, 2.90 mmol, 1.00 equiv),5-cyclopropylisoxazole-3-carboxylic acid (489 mg, 3.19 mmol, 1.10equiv.), dichloromethane (50 mL), and HATU (2.2 g, 5.79 mmol, 1.99equiv). Then TEA (881 mg, 8.71 mmol, 3.00 equiv) was added dropwise. Theresulting solution was stirred for 12 h at room temperature. Thereaction mixture was diluted with 40 mL of DCM and washed with 50 mL ofwater. Then the organic phase was collected and concentrated undervacuum. The residue was purified on a silica gel column (EA: PE=2:3) toget 720 mg (81%) of tert-butyl3-(5-cyclopropylisoxazole-3-carboxamido)azetidine-1-carboxylate ascolorless oil. LCMS (method A, ESI): RT=1.57 min, m/z=252.0 [M-56]⁺.

Step 2: Synthesis ofN-(azetidin-3-yl)-5-cyclopropylisoxazoie-3-carboxamide hydrochloride

Into a 100-mL round-bottom. flask, was placed tert:butyl3-(5-cyclopropylisoxazole-3-carboxatnido)azetidine-1-carboxylate (720mg, 2.34 mmol, 1.00 equiv) and dichloromethane (50 mL). Then hydrogenchloride was introduced into mixture. The resulting solution was stirredfor 5 h at room temperature. The reaction mixture was concentrated. Thisresulted in 560 mg (98%) ofN-(azetidin-3-yl)-5-cyclopropylisoxazole-3-carboxamide hydrochloride asan off-white solid. LCMS (method A, ESI): RT-0.50 min, m/z 208.0 [M+H]⁺.

Step 3: Synthesis of5-cyclopropyl-N-(1-(vinylsulfonyl)azetidin-3-yl)isoxazole-3-carboxamide

Into a 50-mL round-bottom flask was placedN-(azetidin-3-yl)-5-cyclopropylisoxazole-3-carboxamide hydrochloride(100 mg, 0.41 mmol, 1.00 equiv), dichloromethane (20 mL) and TEA (146mg, 1.44 mmol, 3.52 equiv). Then ethenesulfonyl chloride (122 mg, 0.96mmol, 2.35 equiv) was added at room temperature. The resulting solutionwas stirred for 2 h at room temperature. The reaction mixture was washedby 25 mL of water. The organic phase was collected and dried overanhydrous Na₂SO₄. The dried extract was concentrated under vacuum andthe residue purified on a Silica gel column (PE:EA=3:2) to get crudeproduct. Then the resulting product was further purified by prep-HPLCwith the following conditions (Prep-HPLC-025): Column, XBridge Prep C18OBD Column, 5 um, 19*150 mm); mobile phase, WATER WITH 0.05% TFA andMeCN (5.0% MeCN up to 21.0% in 10 min); Detector, UV 254/220 nm. Thisresulted in 35.5 mg (29%) of5-cyclopropyl-N-(1-(vinylsulfonypazetidin-3-yl)isoxazole-3-carboxamideas a white solid. ¹H-NMR (400 MHz, DMSO-d6): δ 9.38 (d, J=12 Hz, 1H),7.04 (q, J=8.4 Hz, 1H), 6.50(s, 1H), 6.32(d, J=6.4 Hz, 1H), 6.21(d,J=8.8 Hz, 1H), 4.69-4.59(m, 1H), 4.04(t, J=12 Hz, 2H), J=10.8 Hz, 2H),2.25-2.16(m, 1H), 1.17-1.04(m, 2H), 0.98-0.89(m, 2H) ppm, LCMS (methodD, ESI): RT−1.61 min, m/z=298.0 [M+H]⁺.

Example 7 Synthesis ofN-(1-(2-chloroacetyl)piperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide(Cpd. No. 4)

Into a 100-mL round-bottom flask was placed5-cyclopropyl-N-(piperidin-4-yl)isoxazole-3-carboxamide hydrochloride(272 mg, 1.00 mmol, 1.00 equiv), dichloromethane (30 mL), and TEA (303mg, 2.99 mmol, 2.99 equiv). Then 2-chloroacetyl chloride (170 mg, 1.51mmol, 1.50 equiv) was added dropwise at 0° C. The resulting solution wasstirred for 12 h at room temperature. The reaction mixture wasconcentrated under vacuum and the residue was purified on a silica gelcolumn with dichloromethane/methanol (10:1) to give 61 mg (20%) ofN-(1-(2-chloroacetyl)piperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamideas a off-white solid. ¹H-NMR (300 MHz, DMSO-d6): δ 8.64 (d, J=9.0 Hz,1H), 6.49 (s, 1H), 4.49-4.20 (m, 3H), 4.13-3.93 (m, 1H), 3.93-3.75 (m,1H), 3.24-3.05 (m, 1H), 2,85-2.65 (m, 1H), 2.25-2.05 (m, 1H), 1.88-1.70(m, 2H), 1.64-1.34 (m, 2H), 1.16-1.02 (m, 2H), 0.97-0.85 (m, 2H)ppm.LCMS (method D, ESI): RT=1.58 min, m/z=312.0 [M+H]⁺.

Example 8 SMYD3 Biochemical Assay General Materials

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), Tris, Tween20,dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well opaque white OptiPlates and SPA beads(Perkin Elmer, catalog #RPNQ001.3) were purchased from PerkinElmer.

Substrates

N-terminally GST-tagged MEKK2 (MAP3K2) protein corresponding toreference sequence AAF63496.3 was purchased from Life Technologies(catalog #PV4010). This protein was expressed in High Five insect cellsand purified to >85% purity. Protein identity was confirmed by MS/MSanalysis after proteolytic digestion. The protein sequence used was:

(SEQ ID No. 1) MAPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRHNQTSLYKKAGTMDDQQALNSIMQDLAVLHKASRPALSLQETRKAKSSSPKKQNDVRVKFEHRGEKRILQFPRPVKLEDLRSKAKIAFGQSMDLHYTNNELVIPLTTQDDLDKALELLDRSIHMKSLKILLVINGSTQATNLEPLPSLEDLDNTVFGAERKKRLSIIGPTSRDRSSPPPGYIPDELHQVARNGSFTSINSEGEFIPESMEQMLDPLSLSSPENSGSGSCPSLDSPLDGESYPKSRMPRAQSYPDNHQEFSDYDNPIFEKFGKGGTYPRRYHVSYHHQEYNDGRKTFPRARRTQGNQLTSPVSFSPTDHSLSTSSGSSIFTPEYDDSRIRRRGSDIDNPTLTVMDISPPSRSPRAPTNWRLGKLLGQGAFGRVYLCYDVDTGRELAVKQVQFDPDSPETSKEVNALECEIQLLKNLLHERIVQYYGCLRDPQEKTLSIFMEYMPGGSIKDQLKAYGALTENVTRKYTRQILEGVHYLHSNMIVHRDIKGANILRDSTGNVKLGDFGASKRLQTICLSGTGMKSVTGTPYWMSPEVISGQGYGRKADIWSVACTVVEMLTEKPPWAEFEAMAAIFKIATQPTNPKLPPHVSDYTRDFLKRIFVEAKLRPSADELLRHMFVHYH.

Molecular Biology

Full-length human SNTYD3 isoform 1 (BAB86333) was inserted into amodified pET21h plasmid containing a His6 tag and TEV and SUMO cleavagesites. Because two common variants of SMYD3 exist in the population,site directed mutagenesis was subsequently performed to change aminoacid 13 from an asparagine to a lysine, resulting in plasmid pEPZ533. Alysine at position 13 conibrms to the more commonly occurring sequence(NP_001161212).

Protein Expression

E. coli (BL21 codonplus RIL strain, Stratagene) were transformed withplasmid pEPZ553 by mixing competent cells and plasmid DNA and incubatingon ice for 30 minutes followed by heat shock at 42° C. for 1 minute andcooling on ice for 2 minutes. Transformed cells were grown and selectedon LB agar with 100 μg/mL ampicillin and 17 μg/mL chloramphenicol at 37°C. overnight. A single clone was used to inoculate 200 mL of LB mediumwith 100 μg/mL ampicillin and 17 μg/mL chloramphenicol and incubated at37° C. on an orbital shaker at 180 rpm. Once in log growth, the culturewas diluted 1:100 into 2 L of LB medium and grown until OD₆₀₀ was about0.3 after which the culture was incubated at 15° C. and 160 rpm. OnceOD₆₀₀ reached about 0.4, IPTG was added to a final concentration of 0.1mM and the cells were grown overnight at 15° C. and 160 rpm. Cells wereharvested by centrifugation at 8000 rpm, for 4 minutes at 4° C. andstored at −80° C. for purification.

Protein Purification

Expressed full-length human His-tagged SMYD3 protein was purified fromcell paste by Nickel affinity chromatography after equilibration of theresin with Buffer A (25 mM Tris, 200 mM NaCl, 5% glycerol, 5 mMβ-mercaptoethanol, pH7.8). The column was washed with Buffer B (Buffer Aplus 20 mM imidazole) and His-tagged SMYD3 was eluted with Buffer C(Buffer A plus 300 mM irnidazole). The His tag, TEV and SUMO cleavagesites were removed generating native SMYD3 by addition of ULP1 proteinat a ratio of 1:200 (ULP1:SMYD3). imidazole was removed by dialysisovernight in Buffer A. The dialyzed solution was applied to a secondNickel column and the native SMYD3 protein was collected from the columnflow-through. The flow-through was dialyzed in Buffer 1) (25 mM Tris, 5%glycerol, 5 mM β-mercaptoethanol, 50 mM NaCl, pH7.8) and ULP1 wasremoved using a Q sepharose fast flow column. SMYD3 was eluted in BufferA and further purified using an S200 size-exclusion column equilibratedwith Buffer A. SMYD3 was concentrated to 2 mg/mL with a final purity of89%.

Predicted Translation:

SMYD3 (Q9H7B4) (SEQ ID No. 2)MEPLKVEKFATAKRGNGLRAVTPLRPGELLFRSDPLAYTVCKGSRGVVCDRCLLGKEKLMRCSQCRVAKYCSAKCQKKAWPDHKRECKCLKSCKPRYPPDSVRLLGRVVFKLMDGAPSESEKLYSFYDLESNINKLTEDKKEGLRQLVMTFQHFMREEIQDASQLPPAFDLFEAFAKVICNSFTICNAEMQEVGVGLYPSISLLNHSCDPNCSIVFNGPHLLLRAVRDIEVGEELTICYLDMLMTSEERRKQLRDQYCFECDCFRCQTQDKDADMLTGDEQVWKEVQESLKKIEELKAHWKWEQVLAMCQAIISSNSERLPDINIYQLKVLDCAMDACINLGLLEEALFYGTRTMEPYRIFFPGSHPVRGVQVMKVGKLQLHQGMFPQAMKNLRLAFDIMRVTHG REHSLIEDLILLLEECDANIRAS.

General Procedure for SMYD3 Enzyme Assays on MEKK2 Protein Substrate

The assays were an performed in a buffer consisting of 25 mM Tris-Cl pH8,0, 1 m.M. TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the dayof use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well whiteopaque OptiPlate using a Bravo automated liquid handling platformoutfitted with a 384-channel head (Agilent Technologies), DMSO (Jul) wasadded to Columns 11, 12, 23. 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of SMYD3, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the SMYD3 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3for 30 min at room temperature, then a cocktail (10 ul) containing SAMand MEKK2 was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: SMYD3 was 0.4nM, ³H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal controlwells was 1 mM, and the DMSO concentration was 2%. The assays werestopped by the addition of non-radiolabeled SAM (10 ul) to a finalconcentration of 100 uM, which dilutes the ³H-SAM to a level where itsincorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 wasdetected using a scintillation proximity assay (SPA). 10 uL of a 1.0mg/mL solution of SPA beads in 0.5 M citric acid was added and theplates centrifuged at 600 rpm fort min to precipitate the radiolabeledMEKK2 onto the SPA beads. The plates were then read in a PerkinElmerTopCount plate reader to measure the quantity of ³H-labeled MEKK2 asdisintegrations per minute (dpm) or alternatively, referred to as countsper minute (cpm).

$\begin{matrix}{\% \mspace{14mu} {inhibition}\mspace{14mu} {calculation}} \\{{\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}}\end{matrix}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

$\begin{matrix}{{Four}\text{-}{parameter}{\mspace{11mu} \;}{IC}\; 50\mspace{14mu} {fit}} & \; \\{Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + {\left( \frac{X}{{IC}_{50}} \right)\mspace{14mu} {Hill}\mspace{14mu} {Coefficient}}} \right.}}} & \;\end{matrix}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

SMYD3 biochemical assay data for representative Compounds of theDisclosure are presented in Table 1 in the column titled “SMYD3 BiochemIC50 (μM).”

Example 9 SMYD3 Cell Assay Trimethyl-MEKK2-in-Cell Western Assay

293T/17 adherent cells were purchased front ATCC (American Type CultureCollection), Manassas, Va., USA. MEM/Glutamax medium, Optimem ReducedSerum medium, penicillin-streptomycin, 0.05% trypsin and 1× D-PBS werepurchased from Life Technologies, Grand Island, N.Y., USA. PBS-10× waspurchased from Ambion, Life Technologies, Grand Island, N.Y., USA. PBSwith Tween 20 (PBST (1.0×)) was purchased from KPL, Gaithersburg, Md.,USA. Tet System FBS- approved FBS US Source was purchased from Cionteeh,Mountain View, Calif., USA. Odyssey blocking buffer, 800CW goatanti-rabbit IgG (IIIl) antibody, 680CW Goat anti-mouse IgG (H+L) andLicor Odyssey infrared scanner were purchased from Licor Biosciences,Lincoln, Nebr., USA. Tri-methyl-Lysine [A260]-MEKK2 antibody, MEKK2 andSMYD3 plasmids were made at Epizyme. Anti-flag monoclonal mouse antibodywas purchased from Sigma, St. Louis, Mo., USA. Methanol was purchasedfrom VWR, Franklin, Mass. USA. 10% Tween 20 was purchased from KPL,Inc., Gaithersburg, Md., USA. Eugene was purchased from Promega,Madison, Wis., USA. The Biotek ELx405 was purchased from BioTek,Winooski, Vt., USA. The multidrop combi was purchased from ThermoScientific, Waltham, Mass., USA.

293T/17 adherent cells were maintained in growth medium (MEM/Glutamaxmedium supplemented with 10% v/v let System FBS and cultured at 37° C.under 5% CO₂.

Cell treatment, In CellWestern (ICV) for Detection ofTrimethyl-Lysine-MEKK2 and MEKK2.

293T/17 cells were seeded in assay medium at a concentration of 33,333cells per cm² in 30 mL medium per T150 flask and incubated at 37° C.under 5% CO₂. Plasmids were prepared for delivery to cells by firstmixing 1350 μL Opti-MEM with Eugene (81 μL) in a sterile Eppendorf andincubated for five minutes at room temperature (RT). MEKK2-flag (13.6ug/T150) MEKK2 p3XFlag-CMV-14 with C-3XFIag and SMYD3 (0.151 ug/T150)SMYD3 p3XPlag-CMV-14 without C-3XFlag plasmids were aliquotted to a 1.7mL sterile microfugc tube. The gene ID for MEKK2 and SMYD3 isNM_006609.3 and Q9H7B4, respectively. Entire volume of Opti-MEM/Fugenemixture was then added to a microfuge tube containing DNA plasmid, mixedand then incubated ×15 minutes at RI. The medium on the 293T/17 cellswas refreshed, and the DNA/Eugene complex is added aseptically to eachflask, rocked gently, and incubated at 37 C for 5 hours. Medium was thenremoved, and cells were washed once with PBS in the flask. Trypsin 0.05%(3 mL) was added and cells incubated for three minutes. Room temperatureMEM+10% Tet system FBS was added and cells were mixed gently, andcounted using the Cells were seeded at 100,000 cells/mL in 50 μL MEM/10%Tet EBS/Pen/Strep to a 384 well black/clear poly-D-lysine coated platecontaining test agent diluted in DMSO. The final top concentration oftest compound was 40 μM. The total concentration of DMSO did not exceed0.2% (v/v). Plates were incubated ×30 minutes at RT in low-airflow area,followed by incubation at 37° C. under 5% CO₂ for 24 hours. Medium wasaspirated from all wells of assay plates prior to fixation andpermeabilization with ice cold (−20° C.) methanol (90 μL/well) for tenminutes. Plates were rinsed with PBS three times on Biolek ELx405. PBSwas removed with a final aspiration, and Odyssey blocking buffer (50μL/well) was added to each well. and incubated for one hour at RT.Primary antibody solution was prepared (anti-trimethyl-MEKK2 at 1:600dilution plus mouse anti-flag antibody at 1:10,000 dilution in diluent(Odyssey Blocking buffer+0.1% Tween 20)) and 20 μL per well wasdispensed using the Multidrop Combi. Assay plates were then sealed withfoil, and incubated overnight at 4° C. Plates were washed five timeswith PBS-Tween (1×) on Biotek ELx405 and blotted on paper towel toremove excess reagent. Detection antibody solution (HZ:Dye 800 CW goatanti-rabbit IgG diluted 1:400 in diluent (Odyssey Blocking buffer+0.1%Tween 20), plus IRD:ye 680CW goat anti-mouse IgG at 1:500 in diluent(Odyssey Blocking buffer+10.1% Tween 20) was added (20 μL(well) andincubated in dark for one hour at RT. Plates were then washed four timeswith PBS-T (1×) on ELx405, A final rinse with water was performed (115×three washes on the ELx405). Plates were then centrifuged upside down,on paper towel, at 200×g to remove excess reagent. Plates were left todry in dark for one hour. The Odyssey Imager was used to measure theintegrated intensity of 700 and 800 wavelengths at resolution of 84 um,medium quality, focus offset 4.0, 700 channel intensity=3.5 to measurethe MEKK2-flag signal, 800 channel intensity 5 to measure theTrimethyl-MEKK2 signal of each well .

Calculations:

First, the ratio for each well was determined by:

$\left( \frac{{Trimethyl}\mspace{14mu} {MEKK}\; 2\mspace{14mu} 800\mspace{14mu} {mn}\mspace{14mu} {value}}{{flag}\mspace{14mu} {tagged}\mspace{14mu} {MEKK}\; 2\mspace{14mu} 700\mspace{14mu} {nm}\mspace{14mu} {value}} \right)$

Each plate included fourteen control wells of DMSO only treatment(Minimum Inhibition) as well as fourteen control wells for maximuminhibition (Background). The average of the ratio values for eachcontrol type was calculated and used to determine the percent inhibitionfor each test well in the plate. Reference compound was serially dilutedtwo-fold in DMSO for a total of nine test concentrations, beginning at40 μM. Percent inhibition was calculated (below).

${{Percent}\mspace{14mu} {Inhibition}} = {100 - \left( {\left( \frac{\begin{matrix}{\left( {{Individual}\mspace{14mu} {Test}\mspace{14mu} {Sample}\mspace{14mu} {Ratio}} \right) -} \\\left( {{Background}\mspace{14mu} {Avg}\mspace{14mu} {Ratio}} \right)\end{matrix}}{\begin{matrix}{\left( {{Minimum}\mspace{14mu} {Inhibition}\mspace{14mu} {Ratio}} \right) -} \\\left( {{Background}\mspace{14mu} {Average}\mspace{14mu} {Ratio}} \right)\end{matrix}} \right)*100} \right)}$

Non-linear regression curves were generated to calculate the IC₅₀ anddose-response relationship using triplicate wells per concentration ofcompound.

SMYD3 cell assay data for representative Compounds of the Disclosure arepresented in Table 1 in the column titled “SMYD3 Cell IC₅₀ (μM),”

Example 10 SMYD2 Biochemical Assay General Materials

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine,Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), andTris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased fromSigma-Aldrich at the highest level of purity possible. ¹H-SAM waspurchase from American Radiolabeled Chemicals with a specific activityof 80 Ci/mrnol. 384-well streptavidin Flashplatcs were purchased fromPerkinElmer.

Substrates

Peptide was synthesized with a N-terminal linker-affinity tag motif anda C-terminal amide cap by 21^(st) Century Biochemicals. The peptide washigh high-perfornance liquid chromatography (HPLC) purified to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was

(SEQ ID NO: 3) ARTKQTARKSTGGKAPRKQLATKAARKSA(K-Biot)-amide.

Production of Recombinant SMYD2 Enzymes for Biochemical Enzyme ActivityAssays

Full length SMYD2 (NP_964582.2) was cloned into a pFastbac-Htb-licvector with an N-terminal His6 tag and FLAG tag, preceded by a TEVprotease cleavage site. The protein was expressed in Sf9 insect cells.Cells were resuspended in lysis buffer (25 mM HEPES-NaOH, pH 7.5, 200 mMNaCl, 5% glycerol, and 5 mM β-ME) and lysed by sonication. The proteinwas purified by Ni-NTA (Qiagen), followed by TEV cleavage to remove theHis6 tag, subtractive Ni-NTA (Qiagen), and gel filtration chromatographyusing an S200 column (GE Healthcare). Purified protein was stored in 20mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM TCEP.

General Procedure for SMYD2 Enzyme Assays on Peptide Substrates

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% Bovine Skin Gelatin, and 0.002% Tween20,prepared on the day of use. Compounds in 100% DMSO (1 ul) were spottedinto a polypropylene 384-well V-bottom plates (Greiner) using aPlatemate Plus outfitted with a 384-channel head (Thermo Scientific),DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for themaximum signal control and 1 ul of SAH, a known product and inhibitor ofSMYD2, was added to columns 11, 12, 23, 24, rows I-P for the minimumsignal control. A cocktail (40 ul) containing the SMYD2 enzyme was addedby Multidrop Combi (Thermo-Fisher). The compounds were allowed toincubate with SMYD2 for 30 min at room temperature, then a cocktail(100) containing ³H-SAM and peptide was added to initiate the reaction(final volume=51 ul). The final concentrations of the components were asfollows: SMYD2 was 1.5 nM. ³H-SAM was 10 nM, and peptide was 60 nM, SAHin the minimum signal control wells was 1000 uM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofnon-radioactive SAM (10 ul) to a final concentration of 600 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed three times with 0.1%Tween20 in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpna) or alternatively, referred to as counts per minute (cpm).

$\begin{matrix}{\% \mspace{14mu} {inhibition}\mspace{14mu} {calculation}} \\{{\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}}\end{matrix}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minandinax are the respective minimum and maximal signal controls.

$\begin{matrix}{{Four}\text{-}{parameter}{\mspace{11mu} \;}{IC}\; 50\mspace{14mu} {fit}} & \; \\{{\% \mspace{14mu} {inhibition}} = {{Bottom} + \frac{{Top} - {Bottom}}{\left( {1 + \left( {{IC}_{50}{\text{/}\lbrack I\rbrack}} \right)^{{Hill}\mspace{11mu} {coefficient}}} \right)}}} & \;\end{matrix}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. I is the compound concentration.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations, and otherparameters without affecting the scope of the invention or anyembodiment thereof.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

All patents and publications cited herein are fully incorporated byreference herein in their entirety.

1. A compound having Formula I:

or a pharmaceutically acceptable salt or hydrate thereof, wherein: R¹ iscyclopropyl; R^(2a), R^(2b), R^(3a), and R^(3b) are selected from thegroup consisting of hydrogen and C₁₋₄ alkyl; X is selected from thegroup consisting of —N(—Y—Z)— and —CH[N(H)—Y—Z]—; Y is selected from thegroup consisting of —C(═O)— and —S(═O)₂—; Z is selected from the groupconsisting of —CH═CH₂, —CH═C(H)CH₂NH₂, —CH═C(H)CH₂N(H)CH₃,—CH═C(H)CH₂N(CH₃)₂, —CH═C(H)CH₂CH₂NH₂, —CH═C(H)CH₂CH₂N(H)CH₃,—CH═C(H)CH₂CH₂N(CH₃)₂, —CH₂Cl, —CH₂Br, and —CH₂I; n is 0 or 1; and m is0 or
 1. 2. A compound of claim 1 having Formula II:

or a pharmaceutically acceptable salt or hydrate thereof.
 3. Thecompound of claim 1, or a pharmaceutically acceptable salt or hydratethereof, wherein R^(2a), R^(2b), R^(3a), and R^(3b) are hydrogen.
 4. Thecompound of claim 1, or a pharmaceutically acceptable salt or hydratethereof, having Formula III:

Formula IV:

Formula V:

or Formula VL

wherein R^(3a) is C₁₋₄ alkyl.
 5. The compound of claim 1, or apharmaceutically acceptable salt or hydrate thereof, having Formula VII:


6. The compound of claim 1, or a pharmaceutically acceptable salt orhydrate thereof, having Formula VIII

or Formula IX:


7. A compound of claim 1 having Formula X:

or a pharmaceutica acce able salt or hydrate thereof.
 8. The compound ofclaim 1, or a pharmaceutically acceptable salt or hydrate thereof,having Formula XI:

or Formula XII


9. The compound of claim 1, or a pharmaceutically acceptable salt orhydrate thereof, wherein Y is —C(═O)—.
 10. The compound of claim 1, or apharmaceutically acceptable salt or hydrate thereof, wherein Y is—S(═O)₂—.
 11. The compound of claim 1, or a pharmaceutically acceptablesalt or hydrate thereof, wherein Z is selected from the group consistingof —CH═CH₂, —CH═C(H)CH₂NH₂, —CH═C(H)CH₂N(H)CH₃, —CH═C(H)CH₂N(CH₃)₂,—CH═C(H)CH₂CH₂NH₂, —CH═C(H)CH₂CH₂N(H)CH₃, —CH═C(H)CH₂CH₂N(CH₃)₂, and—CH₂Cl.
 12. The compound of claim 11, or a pharmaceutically acceptablesalt or hydrate thereof, A.vherein L is selected from the groupconsisting of —CH═CH₂ and —CH₂Cl.
 13. (canceled)
 14. (canceled)
 15. Thecompound of claim 1, or a pharmaceutically acceptable salt or hydratethereof, selected from the group consisting of:(E)-N-(1-((3-aminoprop-1-en-1-yl)sulfonyl)piperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide:(E)-5-cyclopropyl-N-(1-((3-(methylamino)prop-1-en-1-yl)sulfonyl)piperidin-4-yl)isoxazole-3-carboxamide;(E)-5-cyclopropyl-N-(1-(3-(ditnethylamino)prop-1-en-1-)sulfonyl)piperidin-4-yl)isoxazole-3-carboxamide;(E)-N-(1((4-aminobut-1-en-1-yl)sulfonyl)piperidin-4-yl)-5-cyclopropylisoxazole-3-carboxamide;(E)-5-cyclopropyl-N-(1-((4-(methylamino)but-1-en-1-yl)sulfonyl)piperidin-4-yl)isoxazole-3-carboxamide;and(E)-5-cyclopropyl-N-(1-((4-(dimethylamino)but-1-en-1-yl)sulfonyl)piperidin-4-yl)isoxazole-3-carboxamide.16. A pharmaceutical composition comprising the compound of claim 1, ora pharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable carrier.
 17. A method of treating a patientcomprising administering to the patient a therapeutically effectiveamount of claim 1, or a pharmaceutically acceptable salt or hydratethereof, wherein the patient has cancer.
 18. The method of claim 17,wherein the cancer is selected from the group consisting of adrenalcancer, acinic cell carcinoma, acoustic neuroma, acral lentigiousmelanoma, acrospiroma, acute eosinophilic leukemia, acute erythroidleukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia,acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma,adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor,adenosquamous carcinoma, adipose tissue neoplasm, adrenocorticalcarcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia,AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft partsarcoma, ameloblastic fibroma, anaplastic large cell lymphoma,anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma,angiomyolipoma., angiosarcoma, astrocytoma, atypical teratoid rhabdoidtumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocyticleukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer,bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma insitu, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma,chondroma, chordoma, choriocarcinoma, choroid plexus papilloma,clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-celllymphoma, cervical cancer, colorectal cancer, Degos disease,desmoplastic small round cell tumor, diffuse large B-cell lymphoma,dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonalcarcinoma, endocrine gland neoplasm, endodermal sinus tumor,enteropathy-associated T-cell lymphoma, esophageal cancer, fetus infeta, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroidcancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor,gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumorof the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosiscerebri, glucagonoma, gona.dobla.stoma, granulosa cell tumor,gynandroblastoma, gallbladder cancer, gastric cancer, hairy cellleukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma.,hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma,intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna,lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lungcancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma,acute lymphocytic leukemia, acute myelogeous leukemia, chroniclymphocytic leukemia, liver cancer, small cell lung cancer, non-smallcell lung cancer, MALT lymphoma, malignant fibrous histiocytoma,malignant peripheral nerve sheath tumor, malignant triton tumor, mantlecell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia,mediastinal germ cell tumor, medullary carcinoma of the breast,medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkelcell cancer, mesothelioma, metastatic urothelial carcinoma, mixedMullerian tumor, mucinous tumor, multiple myeloma, muscle tissueneoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma.,nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma,neuroma, nodular melanoma, ocular cancer, oligoastrocytoma,oligodendroglioma, oncocytoma, optic nerve sheath meningioma, opticnerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor,papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma,pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma,polyembryoma, precursor T-lymphoblastic lymphoma, primary centralnervous system lymphoma, primary effusion lymphoma, preimary peritonealcancer, prostate cancer, pancreatic cancer, pharyngeal cancer,pseudomyxoma periotonei, renal cell carcinoma, renal medullarycarcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter'stransformation, rectal cancer, sarcoma, Schwannomatosis, seminoma,Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cellcarcinoma, skin cancer, small blue round cell tumors, small cellcarcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinaltumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovialsarcoma, Sezary's disease, small intestine cancer, squamous carcinoma,stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroidcancer, transitional cell carcinoma, throat cancer, urachal cancer,urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer,verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginalcancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms'tumor. 19-24. (canceled)
 25. A kit comprising the compound of claim 1,or a pharmaceutically acceptable salt or hydrate thereof, andinstructions for administering the compound, or a pharmaceuticallyacceptable salt or hydrate thereof, to a patient having cancer.
 26. Thekit of claim 25, wherein the cancer is selected from the groupconsisting of adrenal cancer, acinic cell carcinoma, acoustic neuroma,acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia,acute erythroid leukemia, acute lymphoblastic leukemia, acutemegakaryoblastic leukemia, acute monocytic leukemia, acute promyelocyticleukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoidodontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm,adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressiveNK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma,alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large celllymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma,angiomyolipoma., angiosarcoma, astrocytoma, atypical teratoid rhabdoidtumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocyticleukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer,bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma insitu, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma,chondroma, chordoma, choriocarcinoma, choroid plexus papilloma,clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-celllymphoma, cervical cancer, colorectal cancer, Degos disease,desmoplastic small round cell tumor, diffuse large B-cell lymphoma,dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonalcarcinoma, endocrine gland neoplasm, endodermal sinus tumor,enteropathy-associated T-cell lymphoma, esophageal cancer, fetus infeta, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroidcancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor,gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumorof the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosiscerebri, glucagonoma, gona.dobla.stoma, granulosa cell tumor,gynandroblastoma, gallbladder cancer, gastric cancer, hairy cellleukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma,hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma,intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna,lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lungcancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma,acute lymphocytic leukemia, acute myelogeous leukemia, chroniclymphocytic leukemia, liver cancer, small cell lung cancer, non-smallcell lung cancer, MALT lymphoma, malignant fibrous histiocytoma,malignant peripheral nerve sheath tumor, malignant triton tumor, mantlecell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia,mediastinal germ cell tumor, medullary carcinoma of the breast,medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkelcell cancer, mesothelioma, metastatic urothelial carcinoma, mixedMullerian tumor, mucinous tumor, multiple myeloma, muscle tissueneoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma.,nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma,neuroma, nodular melanoma, ocular cancer, oligoastrocytoma,oligodendroglioma, oncocytoma, optic nerve sheath meningioma, opticnerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor,papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma,pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma,polyembryoma, precursor T-lymphoblastic lymphoma, primary centralnervous system lymphoma, primary effusion lymphoma, preimary peritonealcancer, prostate cancer, pancreatic cancer, pharyngeal cancer,pseudomyxoma periotonei, renal cell carcinoma, renal medullarycarcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter'stransformation, rectal cancer, sarcoma, Schwannomatosis, seminoma,Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cellcarcinoma, skin cancer, small blue round cell tumors, small cellcarcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinaltumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovialsarcoma, Sezary's disease, small intestine cancer, squamous carcinoma,stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroidcancer, transitional cell carcinoma, throat cancer, urachal cancer,urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer,verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginalcancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms'tumor.
 27. A method of treating a SMYD protein mediated disordercomprising administering to a subject in need thereof a compound ofclaim 1, or a pharmaceutically acceptable salt or hydrate thereof in aneffective amount to treat the SMYD protein mediated disorder.